Inactivation of off-taste inducing enzymes

ABSTRACT

The present inventions relates to a method for the production of gellan gum, under mixing conditions, the method comprising a) providing a fermentation broth or other liquid medium containing gellan gum, b) if necessary adjusting the temperature and the pH of the fermentation broth/liquid medium to allow or facilitate enzymatic treatment in step c, c) adding one or more enzymes capable of reducing or abolishing the enzymatic activity of  S. elodea  derived arylsulfatase and/or β-glucuronidase, said one or more enzymes being added in an amount sufficient to reduce or abolish the enzymatic activity of  S. elodea  derived arylsulfatase and/or β-glucuronidase in the broth/liquid medium, and/or treating the broth/liquid medium at a temperature between 90° C. and 125° C. for a period of time sufficient to reduce or abolish the enzymatic activity of  S. elodea  derived arylsulfatase and/or β-glucuronidase in the broth/liquid medium, and d) optionally recovering the gellan gum from the gellan gum containing broth/liquid medium.

FIELD OF THE INVENTION

The present invention relates to reduction of problems with off-tastederived from the activity of impurities in food containing gellan gum.In particular, the present invention relates to means and methods forreducing paracresol formation in gellan containing food products.

BACKGROUND OF THE INVENTION

Gellan gum is a water-soluble polysaccharide produced by the bacteriumSphingomonas paucimobilis formerly named as Sphingomonas elodea orAuromonas elodea or Pseudomonas elodea (Pollock, 1993).

High acyl (HA) Gellan gum is used primarily as a gelling agent. It isable to withstand 120° C. heat. As a food additive, gellan gum is usedas a thickener, emulsifier, and stabilizer. If the gellan gum is added,subsequently heated and cooled, the gellan gum provides a gel of uniquestructure. In manufacture of various dairy products the gellan gum actsas a stabilizer to help formation of a gel. The gellan gum's heatstability and low viscosity at high temperature are especially useful inmanufacturing a product going through heat treatment, such as ultra hightemperature (UHT) or high temperature short time (HTST) treatments.

In foods, gellan gum is used in a variety of products, cf. Duxbury, D D:“Multi-functional gum gets final FDA approval: gellan gum offersformulators low use levels and high versatility”, Food Processing. IssueFebruary 1993. Typical food products incorporating gellan gum includebakery fillings, confections, dairy products, dessert gels, frostings,icings and glazes, jams and jellies, low-fat spreads, microwavablefoods, puddings, sauces, structured foods, and toppings. Gellan gum alsomay be used in canned cat and dog food.

In pharmaceutical products gellan gum can be used to produceeasy-to-swallow solid dosage forms, such as gels and coated tablets, andto modify the rate of release of active ingredients from tablets andcapsules.

However, when adding gellan gum it is almost inevitable that the endproduct is also contaminated with enzymes derived from the bacterialfermentation production of the gum, since the gellan gum is typicallyused in a relatively impure form. These residual enzymes (described asβ-glucuronidase and arylsulphatase) are in turn responsible fordevelopment of an undesirable off-taste in the end-product (oftendescribed as a barn-like or cow-like taste): over time para-cresol(p-cresol) forms as a result of the action of enzymes produced by S.paucimobilis.

Several solutions to this problem have been suggested previously.

In US patent application publication 2006/0003051, it is suggested toproduce a gellan gum composition by a fermentation process comprisinggrowing Sphingomonas elodea in a culture medium, wherein theSphingomonas elodea produces no catalytically active arylsulfatase or nocatalytically active β-glucuronidase or no catalytically activearylsulfatase and no catalytically active β-glucuronidase. In otherwords, US 2006/0003051 utilises genetically modified S. elodea, wherethe genes encoding the two enzymes are suppressed or silenced.

It has also been suggested to pretreat the raw gellan gum compositionwith a denaturing agent prior to combination of the gum with milk. Thismethod is the subject of U.S. Pat. No. 6,663,911.

Finally, US patent application publication 2005/0266138 discloses amethod for preparation of a low calcium sensitive HA gellan gum, where afermentation broth containing the gellan gum is adjusted to a pH about7.5 followed by a subsequent step of pasteurization of the broth.

OBJECT OF THE INVENTION

It is an object of embodiments of the invention to provide alternativemeans and methods for preparation of gellan gum which is, e.g., in turnuseful as a stabilizer in the food industry, namely for the productionof dairy products and beverages.

The invention is particularly useful for dairy products with a desiredlong shelf life at room temperature or above, in that no off-taste isdetected after a long storage time.

SUMMARY OF THE INVENTION

It has been found by the present inventor that the treatment of a gellancontaining broth by a combination of a chelating agent and particularenzymes enables that no development of an off-taste is detected in milkproduct to which the gellan gum isolated from the broth has been added.It has also been found that a thermal treatment of the broth leads tothe reduction of the development of off-taste. Performing both the‘chelatant-enzymes’ treatment and the thermal treatment ensuresexceptionally good results, even in cases where the gellan producingstrain is not genetically modified.

For the good efficiency of the process:

The broth treated is kept warm/hot for the precipitation with alcohol,typically above the gelling temperature. The warm temperature leads thebroth to behave like a liquid and not as a gel and allows an efficientmixing with the alcohol. Thus, it ensures a better quality of theprecipitation and a better yield for the recovery of the fibres.

The precipitation is run continuously, mixing the flow of the broth withthe flow of the alcohol, typically with the help of a pump. Then aresidence time of some minutes allows the fibres to fully precipitate inthe total flow. Doing so, compared with a batch process, the ratio ofalcohol introduced is lower, typically 1/1 in weight, thus significantlyreducing the costs of production.

So, in a first aspect the present invention relates to a method for theproduction of gellan gum, under mixing conditions, the method comprising

-   -   a) providing a fermentation broth or other liquid medium, which        contains gellan gum,    -   b) if necessary adjusting the temperature and the pH of the        fermentation broth or liquid medium to allow enzymatic treatment        in step c,    -   c) adding one or more enzymes capable of reducing or abolishing        the enzymatic activity of S. elodea derived arylsulfatase and/or        β-glucuronidase, said one or more enzymes being added in an        amount sufficient to reduce or abolish the enzymatic activity        of S. elodea derived arylsulfatase and/or β-glucuronidase in the        broth or liquid medium, and/or treating the broth/liquid medium        at a temperature between 90° C. and 125° C. for a period of time        sufficient to reduce or abolish the enzymatic activity of S.        elodea derived arylsulfatase and/or β-glucuronidase in the        broth/liquid medium, and    -   d) optionally recovering the gellan gum from the gellan gum        containing broth/liquid medium.

In a second aspect, the invention relates to preparation of a gellancontaining food product or drug formulation, the method comprisingadmixing gellan gum obtained by the method of the first aspect of thepresent invention with food ingredients or drug components andsubsequently effecting gelation of the gellan gum so as to obtain saidgellan containing food product or drug formulation.

In a third aspect, the present invention relates to a food product or adrug formulation comprising as one ingredient, a gellan gum obtained bythe method of first aspect of the present invention.

DETAILED DISCLOSURE OF THE INVENTION Definitions

An “S. elodea derived arylsulfatase” is an arylsulfatase present in S.elodea or in other gellan gum producing Sphingomonas strains. An S.elodea derived arylsulfatase does hence not need to be produced by S.elodea, but rather by a strain which produces gellan gum. Arylsulfatasesconstitute a group of enzymes active in the hydrolysis of sulfates andthe metabolism of mucopolysaccharides.

An “S. elodea derived β-glucorunidase” is a β-glucorunidase present inS. elodea or in other gellan gum producing Sphingomonas strains. An S.elodea derived β-glucorunidase does hence not need to be produced by S.elodea, but rather by a strain which produces gellan gum.β-glucorunidases are enzymes that attack terminal glycosidic linkages innatural and synthetic glucuronides.

A “food product” is a product intended for oral intake by humans as partof the daily diet. The term covers both solid, semi-solid and liquidproducts, in particular the products discussed above, i.e. the foodproducts discussed in Duxbury, D D: “Multi-functional gum gets final FDAapproval: gellan gum offers formulators low use levels and highversatility”, Food Processing. Issue February 1993. Typical foodproducts thus include bakery fillings, confections, dairy products,dessert gels, frostings, icings and glazes, jams and jellies, low-fatspreads, microwavable foods, puddings, sauces, structured foods, andtoppings.

A “drug formulation” is a mixture between one or more pharmaceuticallyactive ingredients and at least one carrier, vehicle or excipient—drugformulations of particular interest are according to the presentinvention those which are intended for oral intake such as gels andcoated tablets and capsules.

“Dairy products” are defined as foodstuffs produced from milk comingfrom cows or other mammals.

“UHT milk” means milk treated at Ultra High Temperature, typically about135-140° C., during a few seconds.

“HTST milk” means milk treated at High Temperature Short Time, that isto say about 70-75° C. for 15-30 seconds.

“Pasteurization” is defined as a moderate heat treatment not leading tosterilization. The treatment can be e.g. HTST or UHT.

“Diluted milk” means a whole milk diluted with water, lowering the finalamount of proteins to 2% or 1%.

“GC” and “MS” and “SIM” means Gas Chromatography, Mass Spectroscopy,Selective Ion Monitoring, respectively.

A “chelating agent” is in the present context a substance capable ofbinding and stabilizing metallic ions by means of the formation of aninert complex compound or ion in which a metallic atom or ion is boundat two or more points to the substance so as to form a ring structure.

A “protease” (or “proteolytic enzyme) is an enzyme, which degradesproteins and polypeptides by breaking the peptide bonds in said proteinsor enzymes. According to the present invention, suitable proteases arethose capable of degrading S. elodea derived arylsulfatase and/orβ-glucuronidase to such a degree that the enzymatic activity thereof issubstantially reduced or abolished. Preferred proteases can be derivedfrom any source and may be naturally occurring or may be synthetic,semi-synthetic or recombinant analogues of naturally occurringproteases.

“Lysozyme”, also known as muramidase or N-acetylmuramideglycanhydrolase, denotes a family of enzymes (EC 3.2.1.17) whichcatalyze hydrolysis of 1,4-beta-linkages between N-acetylmuramic acidand N-acetyl-D-glucosamine residues in a peptidoglycan and betweenN-acetyl-D-glucosamine residues in chitodextrins.

Specific Embodiments of the Invention First Aspect

In one embodiment of the first aspect of the invention relating topreparation of gellan gum, the mixing conditions may be provided by anymeans conventionally used in the art and are typically provided by astirring power input and/or an aeration flow. Mixing may thus beaccomplished by use of rotors, propellers, pumps, etc. but also bymechanical movement (e.g. via rolling or tipping) of the entirecompartment where the broth is contained—the important goal to achieveis a satisfactory mixing of the contents in the gellan containing brothor liquid medium. Also, the air flow used to maintain a satisfactoryaeration during the process may be used to provide the necessary mixing.

A chelating agent may advantageously be added to the broth/liquid mediumin step a or in step b. Typically, the chelating agent is selected fromcitric acid, oxalic acid, phosphoric acid, DTPA, EDTA, and NTA. In theevent the chelating agent is citric acid, it is preferably added in theform of an about 50% solution to the broth/liquid medium.

In one embodiment (when the method entails addition of enzymes in stepc), the temperature is adjusted to between 20 and 80° C., such asbetween 50 and 60° C., preferably between 53 and 57° C. in step b).Typically, but not necessarily, the temperature is adjusted by directinjection of steam into a fermentation tank comprising the fermentationbroth/liquid medium. It should be noted that the exact choice oftemperature set point typically depends on the characteristics of theenzyme(s) being added—if, for instance, the enzymes exhibit maximumenzymatic activity at temperatures lower than 20 (if they e.g. have beenisolated from organisms that live under cold or cool conditions), thetemperature adjustment will obviously aim at reaching a temperatureinterval at which the enzymes perform satisfactorily.

In another embodiment, step b) further comprises an adjustment of pH.This is also of particular relevance when the method entails addition ofenzymes in step c), so the pH is preferably adjusted to be in the rangeof the optimum activity of said one or more enzymes used in step c.Preferably, the adjustment of pH is done by addition of NaOH, but otheralkaline substances may be utilised.

In a further embodiment, the addition of chelating agent and theadjustment of temperature in steps a) and b) are initiated substantiallysimultaneously.

The one or more enzymes added in step c) are preferably one or both of alysozyme and a protease, such as alcalase.

In step c, the broth/liquid medium is preferably kept at a temperatureabout 55° C. at about pH 8 for a period of time followed by a subsequentadjustment of pH to between 6 and 7.5, preferably between 6.5 and 7. Atemperature “about 55° C.” is intended to denote a temperature not below50° C. and not above 60° C. Normally the temperature will not be below51° C., and it is preferred that the temperature is not below 52° C.; itis more preferred that the temperature is not below 53°, and it isespecially preferred that it is not below 54° C. It is further preferredthat the temperature does not exceed 59° C., and it is preferred that itdoes not exceed 58° C. It is more preferred that the temperature doesnot exceed 57°, and it is especially preferred that it does not exceed56° C. The most preferred temperatures about 55° C. are in the rangebetween 54.5 and 55.5°.

The thermal treatment in step c) is preferably performed at atemperature between 90 and 125° C., and preferably in the temperaturerange between 100 and 120° C. Preferred temperatures are thus about 100,101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114,115, 116, 117, 118, 119 or 120° C.

In certain embodiments of the invention, step c) comprises both additionof the one or more enzymes as well as exercise of the thermal treatment.However, in other embodiments, step c) comprises addition of one or moreenzymes but not thermal treatment, and in other embodiments step c)comprises thermal treatment but not enzyme treatment.

In embodiments where the broth/liquid medium is treated at a temperaturebetween 90° C. and 125° C. in step c, the broth/liquid medium ispreferably cooled down to a temperature which still exceeds the gellingtemperature of the gellan gum (e.g. to about 80° C.) and thenprecipitated, e.g. by addition of an alcohol such as isopropanol. Inpreferred version of this embodiment the precipitation is a continuousprecipitation.

In other, broadly defined embodiments of the first aspect, the inventiongenerally relates to use of lysozyme and/or protease for reducing orabolishing the enzymatic activity of S. elodea derived arylsulfataseand/or β-glucuronidase in a gellan gum containing broth/liquid medium.Similarly, the use of elevated temperature for reducing or abolishingthe enzymatic activity of S. elodea derived arylsulfatase and/orβ-glucuronidase in a gellan gum containing broth/liquid medium alsoconstitute an embodiment of the first aspect, as does use of acombination of a 1) lysozyme and/or protease and 2) elevated temperaturefor reducing or abolishing the enzymatic activity of S. elodea derivedarylsulfatase and/or β-glucuronidase in a gellan gum containingbroth/liquid medium.

These uses falling under the first aspect can also be regarded as use ofelevated temperature and/or of lysozyme and/or of protease in gellan gumproduction to prevent subsequent off-taste in gellan containingproducts.

2^(nd) Aspect

When using the gellan gum obtained by the method of the first aspect ofthe present invention, the gellan gum is generally employed in a mannerknown per se. That is, the gellan gum is admixed with the otheringredients of the product to be produced using process parametersidentical to or equivalent to those utilised when using gellan gumobtained by methods known in the art. The method of the second aspectdoes hence not produce a product which has different characteristics interms of structural differences but rather in a product which exhibits aconsiderable longer shelf-life before any undesirable off-taste developsdue to the activity of S. elodea arylsulfatase and/or β-glucuronidaseactivity.

Hence, in order to exercise the 2^(nd) aspect of the invention, theskilled person may e.g. utilise the guidelines and recommendationsprovided in Duxbury et al. (supra); Giavasis I, Harvey L M, McNeil B:“Gellan Gum,” Crit Rev Biotechnol, 20(3), 177-211,2000; Miyazaki S,Aoyama H, Kawasaki N, et al.: “In situ gelling formulations as Gellanvehicles for oral delivery [J],” J Controlled Release, 60 (3), 287,1999; Morris V J: “Bacterial polysaccharides, in Food polysaccharidesand their application”, Stephen A M, Editor, Marcel Dekker, New York,341-375, 1995.

3^(rd) Aspect

Closely related to the above aspects is the 3^(rd) aspect, which relatesto products (food products or drug formulations) containing the gellangum obtained by the method of the invention.

Food products which contain the gellan gums are typically selected fromthe group consisting of bakery fillings, confections, dairy products,dessert gels, frostings, icings and glazes, jams and jellies, low-fatspreads, microwavable foods, puddings, sauces, structured foods, andtoppings, whereas drug formulations typically are selected from thegroup consisting of a gel, a tablet, and a capsule. In both cases, thegellan gum obtained by the method of the invention is preferably presentin an amount effective to function as a thickener and/or emulsifierand/or stabiliser.

EXAMPLES Materials and Methods 1. ‘Chelatant-protease’ TreatmentProtocol

During the fermentation, the pH is adjusted between 6.4 and 7 dependingon the metabolism. When fermentation is completed and because there isno more sugar to metabolize, the respiratory activity decreases and thepH, which is no longer controlled, increases (because of the release ofdissolved CO₂).

Then the treatment, under mixing conditions, is:

The broth is heated by a direct injection of steam in the fermenteruntil about 55° C. is reached and air is fed at the same time to helpmix.

As soon as the heating is started, 160 kg of a 50% solution of citricacid is added with help of a pump. The pH should drop to about 5.

Heating is stopped when T=55° C.

Once the temperature has reached 55° C., the pH is increased to about8.0 with a base.

The pH is kept at about 8.0.

The following enzymes are added:

-   -   Lysozyme at about 230 ppm    -   Protease at about 1000 ppm

The mixture is kept at about 55° C. for about 1 hour while the pH ismaintained at about 8.0.

The pH is then lowered to about 7 using, e.g., H₂SO₄.

The broth is transferred to a holding tank while adding 10% ofisopropanol.

Mixing conditions are adjusted by the stirring power input and/oraeration flow.

A number of experiments where performed with different proteases andwith modified versions of the ‘chelatant-protease’ treatment:

Experiments 56-6 and 58-B: the protocol conditions, but Protease wastype I (Alcalase 2.4 L) at 500 ppm.

Experiment 72-3: the protocol conditions but with Protease type II(Protex 6L).

Experiments 72-4 and 72-7: the protocol conditions, but with Proteasetype III (Proteinase T) at 2000 ppm.

Experiment 72-5: the protocol conditions, but with Protease type IV(Multifect) at 2000 ppm.

Experiments 72-6: the protocol conditions, but Protease was type I(Alcalase 2.4 L) at 1000 ppm.

Experiments 74-2 and 80-A: the protocol conditions with Protease type II(Protex 6L).

Experiment 80-B: the protocol conditions, but with sodium citrateinstead of citric acid and no pH control.

2. Thermal Treatment Protocol

Following the Chelatant-protease treatment, the broth undergoes heattreatment as follows:

The pH is adjusted between 6 and 7.5, and preferably at 6.5.

The temperature is adjusted between 90° C. and 125° C., preferablybetween 100-110° C.

The residence time is at least 15 minutes.

In the following experiments, the conditions applied are as follow:

Experiment 72-1: 100° C., pH=7, residence time 15 min.

Experiment 72-2: 120° C., pH=7, residence time 20 min.

Experiments 74-2 and 74-3: 100° C., pH=6.5, residence time 20 min.

Experiments 72-3, 72-4, 80-A, 80-B: 100° C., pH=6.5, residence time 15min.

After the thermal treatment, the broth is cooled down to a temperaturenot lower than the gelling temperature, e.g. at about 80° C. Then it isprecipitated in a continuous process, by addition of isopropanol.

3. NaOH Treatment Protocol

U.S. Pat. No. 6,663,911 describes different protocols using an alkali oran oxidising agent to treat a gellan broth.

In order to compare our invention to the one described in the U.S. Pat.No. 6,663,911, we applied the protocols described in the U.S. Pat. No.6,663,911 to our own broth. The conditions are as follows:

Experiments 56-8, 72-8, and 74-3: 1 g NaOH/10 litres of broth, 50° C.,3h, pH not controlled.

Experiments 56-9: NaOH added up to increase the pH=9 (about 2 g/10litres of broth), then, 50° C., 3h, pH not controlled.

4. NaOCl Treatment Protocol

U.S. Pat. No. 6,663,911 describes different protocols using an alkali oran oxidising agent to treat a gellan broth.

In order to compare our invention to the one described in the U.S. Pat.No. 6,663,911, we applied the protocols described in the U.S. Pat. No.6,663,911 to our own broth. The conditions are as follow:

Experiments 56-7, and 72-10: 1000 ppm NaOCl, 50° C., 3h, pH notcontrolled.

5. Preparation of Samples in UHT Milk

Trials in UHT milk have to be run to determine if the final gellanproduct is suitable for an application in milk.

A gellan gum known in the market such as KELCOGEL LT100 from CP-Kelco,was used as a positive control, in the following protocol.

Ingredients in %:

Cream 35% fat: 8.45

Saccharose: 5

Gellan gum: 0.09 to 0.15

Skimmed milk (pasteurised): 86.4 to 86.46

Protocol for UHT Plates Heat Exchanger:

Blend all ingredients.

Preheating at 70° C.

Homogenize in 2 steps up to 100 bar at 70° C.

Heating at 90° C./holding time 30 s.

UHT treatment at 140° C. for 3 seconds.

Cooling down to 10° C. and fill in the containers

Storage at two different temperatures: 5° C. and 20° C.

6. Assays for Determination of Para-cresol Content

p-Cresol concentrations have been determined by Gas Chromatography:

-   To 10 g of sample add 1 ml 2N HCl, 5 ml demineralised water and    internal standard corresponding to approx. 100 ppb. The internal    standard is ethyl anisate.

Extract with 10 ml diethylether using magnetic stirring for 30 minutes.

-   The ether extract is analysed by GC/MS/SIM.-   Each sample is analysed in duplicate and standard addition of    p-cresol at a level of 50 and 100 ppb is performed to several    samples to verify method performance.-   Calibration is performed in the range 5 to 250 ppb (external    calibration in diethyl ether).

Results

1. Evaluation of Series 1 after a 4-week storage.

Storage at 4° C.:

Exp. 56-4 (no treatments): off-taste 55 ppb of p-cresol Exp. 56-6(Chelatant/protease): no off-taste 12 ppb of p-cresol Exp. 56-7 (NaOCl1000 ppm): bitter off-taste Exp. 56-8 (NaOH 1 g/kg): bitter off-tasteExp. 56-9 (NaOH for pH = 9): slight off-taste

Storage at 20° C.:

Exp. 56-6 (‘Chelatant/protease): no off-taste 9 ppb of p-cresol Exp.56-7 (NaOCl 1000 ppm): hot milk taste Exp. 56-8 (NaOH 1 g/kg): off-tasteExp. 56-9 (NaOH for pH = 9): fermented milk taste

Positive control

At 20° C.: strong cow-like off taste 160 ppb of p-cresol At 4° C.:cow-like off taste  85 ppb of p-cresol2. Evaluation of Series 2 after a 4-week Storage

Storage at 20° C.:

-   Exp. 58-A (no treatment): off-taste-   Exp. 58-B (Chelatant/proteasel): milky, no off-taste

Positive control strong cowy off-taste, bitterness

3. Evaluation of Series 3 after a Storage Time up to 16 Weeks

For the following experiments, different protocols have been applied.

Exp. 72-1 (thermal treatment at 100° C., 15 min)

Exp. 72-2 (thermal treatment at 120° C., 20 min)

Exp. 72-3 (Chelatant/protease treatment+thermal treatment at 100° C., 15min)

Exp. 72-4 (Chelatant/protease treatment+thermal treatment at 100° C., 15min)

Exp. 72-5 (Chelatant/protease treatment)

Exp. 72-6 (Chelatant/protease treatment)

Exp. 72-7 (Chelatant/protease treatment)

Exp. 72-8 (NaOH, 1 g/l, at 50° C.)

Exp. 72-10 (NaOCl, 1000 ppm, at 50° C.)

Exp. 74-2 (Chelatant/protease treatment+thermal treatment at 100° C., 20min)

Exp. 74-3 (NaOH+thermal treatment at 100° C., 20 min)

Exp. 80-A (Chelatant/protease treatment+thermal treatment at 100° C., 15min)

Exp. 80-B (Chelatant/protease treatment, replacing citric acid by sodiumcitrate, no pH control+thermal treatment at 100° C., 15 min)

Exp.1201, Exp. 1202, Exp. 1301, Exp. 1401, Exp. 1402

For all the previous 5 experiments, at industrial scale, we applied‘Chelatant/protease’ treatment+thermal treatment at 100° C., 20 min.

Evaluations for the off-taste for the series 3 are shown in thefollowing Table:

Storage at 20° C. Dosage* Evaluation of off-taste after # of weeks:p-cresol Reference (%) 4 8 16 (ppb) 72-1 0.15 barn-like− — — — 72-2 0.15no no — 18 72-3 0.15 no no no 16 72-4 0.09 no no no <10 72-5 0.15 no no— — 72-6 0.11 no no no <10 72-7 0.14 no no no <10 72-8 0.15 barn-likebarn-like — — 72-10 0.13 no off-taste barn-like− — positive 0.15barn-like+ barn-like++ barn-like+++ 90 control Milk — no no no —(negative control) 74-2 0.12 no no no <10 74-3 0.15 no off-tasteoff-taste <10 but no but no p-cresol p-cresol positive 0.15 barn-likebarn-like+ barn-like++ 60 control Milk — no no no — (negative control)80-A 0.15 no no no <10 80-B 0.15 no no no 14 positive 0.15 barn-like+barn-like++ barn-like+++ 100 control Milk — no no no — 1201 0.15 no nono <10 1202 0.15 no no no <10 1301 0.15 no no no <10 1401 0.15 no no no<10 1402 0.15 no no no <10 positive 0.15 barn-like barn-like+barn-like++ 80 control Milk — no no no — (negative control) *Dosage isthe amount of gellan gum in the milk. For the evaluation, + means‘detected’, ++ means ‘significant’, +++ means ‘strong’.

Discussion

It is confirmed that the ‘chelatant/protease’ protocol alone or inaddition with a thermal treatment leads to very good results.

The protocols with NaOH or NaOCl disclosed in U.S. Pat. No. 6,663,911can lead to an off-taste; this off-taste is different from the off-tastecharacterising the presence of p-cresol.

Concerning the thermal treatment as sole treatment, it seems that thetemperature is a sensitive parameter. Two trials support this point:

-   -   Exp. 72-1 with 100° C. led to a p-cresol off-taste.    -   Exp. 72-2 with 120° C. led to no p-cresol off-taste.

A pasteurization (understood in the state of the art as being performedat a moderate temperature and/or duration) would therefore seem to beinsufficient to denature the p-cresol causing enzymes.

It is therefore preferable to use a thermal treatment, with temperatureabove 100° C. and a long duration of about 15 minutes.

Also, in addition to the trials described above, a trial has been run inretorted milk, which means “99.78% milk+0.2% sucrose+0.02% gellan gum”sterilized in cans at 121° C. for 15min, in an autoclave. No p-cresol isdetected after 6-weeks of storage at either 20° C. or 40° C.

In the enzymatic reaction releasing p-cresol, because the positivecontrol leads to a quite high concentration in p-cresol, this indicatesthat there is no limitation by the precursors present in the milk. Thus,it can be assumed that the enzymes are the limiting factor. So, theproduction of p-cresol will directly depend on the percentage of gellanin the milk. A test run at 0.15% can be considered as an acceleratedtest of a final 0.05% recipe. If no p-cresol detected after 16 weekswith a 0.15% dosage, it can be assumed that no p-cresol would bedetected after 48 weeks with a 0.05% dosage.

Conclusions

There is no doubt that a ‘chelatant/protease’ treatment applied to thegellan broth results in no development of a p-cresol off-taste in UHTmilk during a long storage duration.

Following some trials and also considering the results in retorted milk,we can conclude that a thermal treatment is also able to denature theenzymes responsible of the p-cresol production.

Therefore, both these approaches in isolation are useful according tothe invention for reducing/abolishing off-taste problems in gellan gumcontaining products. However, it appears that optimum results areobtained when combining a “chelatant/protease” treatment in combinationwith a thermal treatment.

1.-28. (canceled)
 29. A method for a production of gellan gum, undermixing conditions, the method comprising: providing a liquid mediumcontaining the gellan gum; adjusting a temperature and a pH of theliquid medium to facilitate enzymatic treatment; applying the enzymatictreatment, wherein the enzymatic treatment comprises one or more of: 1)adding one or more enzymes capable of reducing or abolishing theenzymatic activity of one or more of S. elodea derived arylsulfatase orβ-glucuronidase, wherein the one or more enzymes are added in an amountsufficient to reduce or abolish the enzymatic activity of the one ormore of the S. elodea derived arylsulfatase or the β-glucuronidase inthe liquid medium, and 2) treating the liquid medium at a temperaturebetween 90° C. and 125° C. for a period of time sufficient to reduce orabolish the enzymatic activity of the one or more of the S. elodeaderived arylsulfatase or the β-glucuronidase; and recovering the gellangum from the liquid medium.
 30. The method of claim 29, wherein thetemperature is adjusted to between 53° C. and 57° C. when adjusting thetemperature and the pH of the liquid medium to facilitate enzymatictreatment.
 31. The method of claim 29, wherein the pH is adjusted to bein a range of an optimum activity of the one or more enzymes appliedduring the enzymatic treatment.
 32. The method of claim 29, wherein theliquid medium is kept at about 55° C. and at about the pH of 8.0 for aperiod of time followed by adjustment of the pH to between 6.5 and 7.0.33. The method of claim 29, wherein mixing conditions are provided byone or more of a stiffing power input or an aeration flow.
 34. Themethod of claim 29, wherein a chelating agent is added to the liquidmedium before or during the adjusting of the temperature and the pH ofthe liquid medium.
 35. The method of claim 34, wherein the chelatingagent is selected from the group comprising one or more of citric acid,oxalic acid, phosphoric acid, DTPA, EDTA, or NTA.
 36. The method ofclaim 35, wherein the citric acid is added in the form of about 50%solution to the liquid medium.
 37. The method of claim 29, wherein therecovered gellan gum is admixed with food ingredients to effect gelationof the gellan gum to produce a gellan-containing food.
 38. The method ofclaim 29, wherein the recovered gellan gum is admixed with drugcomponents to effect gelation of the gellan gum to produce agellan-containing drug composition suitable for oral use.
 39. The methodof claim 37, wherein the gellan-containing food comprises bakeryfillings, confections, dairy products, dessert gels, frostings, icings,glazes, jams, jellies, low-fat spreads, microwavable foods, puddings,sauces, structured foods, and toppings.
 40. The method of claim 38,wherein the drug formulation is selected from the group comprising agel, a tablet, and a capsule.
 41. The method of claim 38, wherein thegellan gum in the drug formulation is present in an amount effective tofunction as one or more of a thickener, emulsifier or stabilizer. 42.The method of claim 37, wherein the gellan gum in the gellan-containingfood is present in an amount effective to function as one or more of athickener, emulsifier or stabilizer.
 43. The method of claim 29, whereinafter the liquid medium is treated at the temperature between 90° C. and125° C., the liquid medium is cooled down to the temperature of about80° C. and then precipitated by addition of an alcohol.
 44. The methodof claim 43, wherein the precipitation is a continuous precipitation.45. A method for a production of gellan gum, under mixing conditions,the method comprising: providing a liquid medium containing the gellangum; adjusting a temperature and a pH of the liquid medium to facilitateenzymatic treatment; applying the enzymatic treatment, wherein theenzymatic treatment comprises adding one or more of a lysozyme or aprotease capable of reducing or abolishing the enzymatic activity of oneor more of S. elodea derived arylsulfatase or β-glucuronidase, whereinthe one or more of the lysozyme or the protease are added in an amountsufficient to reduce or abolish the enzymatic activity of the one ormore of the S. elodea derived arylsulfatase or the β-glucuronidase inthe liquid medium; and recovering the gellan gum from the liquid medium.46. The method of claim 45, wherein the enzymatic treatment comprisesadding an alcalase that confers both lysozyme and protease enzymaticactivity.
 47. A method for a production of gellan gum, under mixingconditions, the method comprising: providing a liquid medium containingthe gellan gum, wherein the gellan gum comprises one or more of a S.elodea derived arylsulfatase or a β-glucuronidase; treating the liquidmedium at a temperature between 90° C. and 125° C. for a period of timesufficient to reduce or abolish enzymatic activity of the one or more ofthe S. elodea derived arylsulfatase or the β-glucuronidase; andrecovering the gellan gum from the liquid medium.
 48. The method ofclaim 47, wherein the liquid medium is treated at the temperaturebetween 100° C. and 120° C.